In mammals, life begins at fertilization when the sperm interacts with the oocyte to trigger a series of intracellular Ca2+ oscillations that persists for several hours. This prolonged period of repetitive Ca2+ pulses triggers the developmental program by stimulating the enzymatic machinery involved in the cell division cycle. Further changes in intracellular Ca2+ are not observed until the one cell embryo is ready to divide, when a spontaneous Ca2+ transient triggers cleavage to form two daughter cells. The early steps that lead to the rises in calcium and oocyte activation at fertilization were unknown but of great interest, particularly with the advent of in vitro fertilization techniques, and whole-animal cloning by nuclear transfer. This calcium rise is required for oocyte activation and the subsequent events of development in eggs or oocytes of all species.
The meiotic division of mammalian oocytes begins with one primary germ cell (oocyte), which gives rise to only one mature ovum (egg). In normal mammalian development, oocytes become developmentally arrested in the ovaries at the germinal vesicle stage in prophase of the first meiotic division. Upon appropriate stimulation, meiosis resumes, the germinal vesicle breaks down, and the first meiotic division is completed with the extrusion of a diploid set of chromosomes into the first polar body, another diploid set of chromosomes remaining within the cytoplasm of the oocyte. The oocyte then becomes arrested at metaphase of the second meiosis (xe2x80x9cMet IIxe2x80x9d). Met II oocytes (mature oocytes) can then be ovulated and fertilized.
Once fertilized, the activated oocyte completes the second meiotic division with the extrusion of a haploid set of chromosomes into the second polar body, male and female pronuclei are formed, and DNA replication is initiated in the pronuclei. The male and female pronuclei then fuse together, allowing their chromosomes to mingle. Equal segregation of the genetic material occurs by mitosis and the zygote cleaves to form two daughter blastomeres. The embryo continues to develop by undergoing a series of mitotic divisions before differentiating into specific cells, resulting in the organization of tissues and organs. This developmental program ensures the successful transition from oocyte to offspring.
When this process is defective, for example the inability of sperm to fertilize the egg, technical assistance has been sought. Since the first application of in vitro fertilization (IVF) in humans, the number of patients using assisted reproductive technologies (ART) has increased tremendously in number and technological spectrum. Infertility affects approximately one out of every six couples in the United States who desire children. For about 30% of couples male factor infertility will be the sole cause. An additional 20% will also have female factor infertility as an additional contributory factor. Therefore, a large percentage of infertility cases have a contributory male component.
Some options for dealing with male infertility include intracytoplasmic sperm injection (ICSI), which may use pre-ejaculate sperm for fertilization. The use of epididymal spermatozoa in assisted reproduction (ART) permits fertility in men with surgically irremediable obstructive azoospermia. However, when used for conventional IVF (sperm/oocyte co-culture), epididymal spermatozoa show reduced fertilization and pregnancy rates due to their functional immaturity. Part of this maturation occurs as the sperm cells move through the ducts of the male genital tract where fluid from the prostate gland and the seminal vesicles mix with the cells, forming the semen. Nevertheless, freshly ejaculated sperm are not yet capable of fertilizing an egg. The sperm must also go through the process of capacitation in the female genital tract that leads to release of enzymes that ultimately help the sperm penetrate an egg. Therefore, epididymal spermatocytes have an improved chance of fertility when combined with intracytoplasmic sperm injection (ICSI).
In addition to ICSI, nuclear transfer may have enormous applications in the fields of agriculture and biomedicine. This is especially true if a cell line that has been transformed can be used as a source of nuclei for the nuclear transfer and thus give rise to the cloning of an individual with an identical genetic composition as the transferred nucleus. One major aspect of the nuclear transfer and ICSI procedures is that of oocyte activation. Without oocyte activation the transferred nucleus would never progress to the first interphase. The inability to obtain development after artificial activation of oocytes has been a limiting factor in the application of these technologies. It is therefore of utmost importance that the oocyte be activated in a fashion that closely mimics the natural process which occurs at fertilization.
Improved methods of obtaining or preventing oocyte activation may find use with in vitro fertilization, nuclear transfer, contraception, and other reproductive technologies. The present invention addresses this issue.
Relevant Literature:
The role of calcium in biological signaling is reviewed by Berridge et al. (1998) Nature 395:645. The biology of egg activation is reviewed by Epel, in Handbook of Physiology (eds. Hoffman and Jamieson) 859-884 (Oxford Univ. Press, N.Y., 1997); and Stricker (1999) Dev. Biol. 211:157-176. Methods for parthenogenic oocyte activation are described in U.S. Pat. No. 6,077,710, Susko-Parrish et al.
The use of nitric oxide synthase modulators in enhancing implantation of embryos is discussed in U.S. Pat. No. 6,040,340, Chwalisz et al.
Methods are provided for the modulation of oocyte activation through modulation of nitric oxide levels. The invention is based on the finding that nitric oxide synthase and nitric oxide related bioactivity are necessary and sufficient for activation of an oocyte during the process of fertilization.
In one embodiment of the invention, oocyte activation is enhanced by the addition of nitric oxide synthase, nitric oxide synthase enhancers, nitric oxide donors, or other compounds that modulate the levels of nitric oxide in the oocyte during fertilization. The oocyte activation finds particular use in conjunction with artificial reproduction technologies, such as nuclear transfer, intracytoplasmic sperm injection, in vitro fertilization, and the like.
In another embodiment of the invention, the prevention of nitrosation of the oocyte, e.g. through the administration of NOS inhibitors, is used to block oocyte activation, where contraception is desired.